Resin-metal composite, labeling substance, immunoassay method, immunoassay reagent, method for measuring analyte, analyte measurement kit, and lateral-flow chromatographic test strip

ABSTRACT

A resin-metal composite  100  includes resin particles  10  and metal particles  20 . The metal particles  20  are dispersed or immobilized on the resin particles  10 , and portions of the metal particles  20  are three-dimensionally distributed in a surface section  60  of the resin particles  10 . The metal particles  20  include encased metal particles  30  completely encased in the resin particles  10 , partially exposed metal particles  40  having a portion embedded in a resin particle  10  and a portion exposed from the resin particle  10 , and surface-adsorbed metal particles  50  absorbed on the surface of a resin particle  10.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a resin-metal composite applicable to,for example, markers and so on applicable to immunoassay, a markerutilizing same, an immunoassay method, an immunoassay reagent, a methodfor measuring analyte, an analyte measurement kit, and a lateral-flowchromatographic test strip.

2. Description of Related Art

Innumerable chemical substances exist in an organism, so the qualitativeand quantitative analysis of specific trace components in the organismis an extremely important technique. In fields, such as medical care,pharmacy, health food, biotechnology and environment, drugs and foodhaving effects only on specific parts (chemical substances) inorganisms, analysis devices and diagnostic drugs and the like detectingslight changes in organisms develop along with the technique.

One of the analysis techniques is immunoassay. This technique is alsocalled immunological assay, which is a method that qualitatively andquantitatively analyzes trace components by utilizing antigen-antibodyspecific reaction as one of immunoreactions. Because of high sensitivityor reaction selectivity, the antigen-antibody reaction is widely appliedin the fields. Various assay methods exist in immunoassay according toassay principles. Examples are listed as follows: enzyme immunoassay(EIA), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA),fluorescence immunoassay (FIA), agglutination of latex and so on (lateximmunoassay (LIA), particle agglutination (PA)), immunochromatography(ICA), hemagglutination (HA) and hemagglutination inhibition (HI).Moreover, besides immunoassay, there are physical and chemical assays,biological assay and so on.

Immunoassay qualitatively or quantitatively assays an antigen or anantibody according to change in the reaction between the antigen and theantibody for forming the composite (concentration change of the antigen,the antibody or a composite). When these are assayed, the antibody, theantigen or the composite are bound with a marker, and thereby assaysensitivity is increased.

Therefore, it can be said that the marking capability of the marker is asignificant factor affecting assay capability in immunoassay. In theforegoing illustrated immunoassays, erythrocytes (the case of HA), latexparticles (the case of LIA), fluorochromes (the case of FIA),radioactive elements (the case of RIA), enzymes (the case of EIA) andchemiluminescent substances (the case of CLIA) and so on can be adoptedas markers.

However, when colored microparticles are used as a marker, assay can bedetermined visually without using a special analysis device, so it canbe expected that simpler assay may be realized. As such coloredmicroparticles, examples are listed as follows: colloidal particles ofmetals and metal oxides and latex particles colored by utilizing apigment (patent document 1, patent document 4, etc.). However, becausethe color of the colloidal particles is determined according to particlesizes and preparation conditions, there exists a problem that it is hardto obtain a desired bright rich color, that is, visual recognizabilityis insufficient.

In addition, the colored latex particles have the problem of lowcoloring effect of the utilized pigment and insufficient visualdeterminability. Furthermore, if the coloring amount of the pigment isincreased in order to solve the problem, then the pigment covers thesurface of the latex, the original surface state of the latex particlesis impaired, and as a result, there exists a problem that it is hard tobind the antigen or the antibody. In addition, there also exist thefollowing problems: blockage in pores of a chromatographic medium suchas a membrane filter, or nonspecific agglutination produced by the latexparticles or uncertain association between rich coloration resultingfrom the increase in the coloring additive of the pigment and anincrease in performance.

In order to improve the visual recognizability of the marker, followingimmunochromatography is disclosed: after an antibody (marked antibody)bound with a marker react with an antigen to form a composite, othermetals modify the marker, and thereby the assay sensitivity of themarker is increased (patent document 2 and patent document 5). However,in the method, operation is complex, and it is hard to achieve a stableincrease. In addition, it is considered that assay cost is consumed dueto the fact that a special device is required, so applicable purposesand application environment are limited.

In addition, a coloring latex containing gold nanoparticles bound withthe surfaces of polymeric latex particles (patent document 3) isdisclosed.

By binding the surfaces of the polymeric latex particles with the goldnanoparticles, the gold nanoparticles as colorant itself can help toincrease visual determinability or assay sensitivity. On the other hand,the gold nanoparticles itself are excellent for the binding of anantigen or an antibody, so, even if the gold nanoparticles are bounduntil a degree of sufficiently rich color, an enough amount of antigenor antibody can be bound.

γrays irradiate a dispersion of styrene-acrylic acid copolymer latex anda precursor of gold nanoparticles (i.e. HAuCl), so that the surface ofthe latex is bound with the gold nanoparticles, and thereby the coloringlatex is formed. However, since the gold nanoparticles are only boundwith the surface of the latex, not only is the quantity of carried goldparticles manifesting surface plasmon absorption limited, but also thegold nanoparticles can easily come off. As a result, the visualrecognizability or sensitivity of it as an immunoassay reagent may notbe sufficient. In addition, because of the irradiation ofelectromagnetic radioactive rays such as γrays, the latex may beinjured. Further, although the description of patent document 3discloses preferred ranges of latex size or gold nanoparticle size,whether it has been verified within these preferred ranges in theembodiments is not clear, so a specified basis of preferred ranges doesnot exist.

In addition, patent document 4 discloses a metal gold-coated polymerlatex particle, and suggests the application of a reagent applicable tomicroscopic examination and immunoassay.

However, the material or particle size of the metal gold-coated polymerlatex particle is not disclosed. Further, the effect of it as a reagentapplicable to immunoassay has not been verified. Therefore, the effectof it as a reagent in metal gold and polymer latex particles is notclear.

In addition, non-patent document 1 discloses a microgel which isprepared by carrying gold nanoparticles on poly-2-vinylpyridine latexparticles, and determines the pH responsibility of the particle size ofthe microgel according to change in the behavior of localized surfaceplasmon absorption of the gold nanoparticles. However, the microgelcarries the gold nanoparticles on a single layer near the surfaces ofthe latex particles. Therefore, it can be considered that the quantityof carried the gold nanoparticles is low, and as a result, a rich colorwhich is effective for immunoassay cannot be obtained. In addition,research on the material, structure and composition of the microgel hasnot been made, so the effect of specific applications, such as animmunoassay reagent, is not clear.

According to what is mentioned above, although the latex particle boundor coated with the gold nanoparticle is expected as an immunoassayreagent, in the prior art, durability or visual recognizability is notsufficient. In addition, even if visual recognizability is high,applicable purposes and application environment are limited.

DOCUMENTS OF THE PRIOR ART Patent Documents

-   Patent Document 1: Japanese Unexamined Patent Application    Publication No. Hei-5-10950-   Patent Document 2: Japanese Unexamined Patent Application    Publication No. 2011-117906-   Patent Document 3: Japanese Unexamined Patent Application    Publication No. 2009-168495-   Patent Document 4: Japanese Unexamined Patent Application    Publication No. Hei-3-206959-   Patent Document 5: Japanese Unexamined Patent Application    Publication No. 2009-192270

Non-Patent Document

-   Non-patent Document 1: K. Akamatsu, M. Shimada, T. Tsuruoka, H.    Nawafune, S. Fujii and Y. Nakamura, Langmuir, 2010, 26, 1254-1259

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

The present invention is directed to provide a resin-metal compositewhich is excellent in durability and visual recognizability and canrealize high-sensitivity determination without requiring the addition ofa special device or operation steps in applications such as a materialfor immunoassay.

Means for Solving the Problem

As the result of the research effort of the inventor and the others, itis discovered that a resin-metal composite with a specific structure cansolve the problem, and thereby the present invention is achieved.

That is, the resin-metal composite of the present invention comprises astructure which is formed by immobilizing a plurality of metal particleson a resin particle, and is characterized in that the plurality of metalparticles includes:

first particles, having a portion exposed from the resin particle; and

second particles, completely encased in the resin particle; and

among the first particles and the second particles, at least portions ofthe particles are three-dimensionally distributed in a surface sectionof the resin particle.

In the resin-metal composite of the present invention, relative to theweight of the resin-metal composite, the quantity of the carried metalparticles can be within a range from 5 wt % (percentage by weight) to 70wt %.

In the resin-metal composite of the present invention, 60 wt % to 100 wt% of the metal particles can exist in the surface section, and 5 wt % to90 wt % of the metal particles existing in the surface section can bethe first particles.

In the resin-metal composite of the present invention, the resinparticle can be a polymer particle which is provided with a substituentgroup capable of adsorbing metal ions in the structure.

In the resin-metal composite of the present invention, the averageparticle size of the metal particles can be within a range from 1 nm to80 nm.

The average particle size of the resin-metal composite of the presentinvention can be within a range from 100 nm to 1000 nm.

In the resin-metal composite of the present invention, the metalparticles can be gold particles.

A marker of the present invention comprises any one of the resin-metalcomposites.

The marker of the present invention can be used in adsorbing an antigenor an antibody on the surface of the resin-metal composite.

An immunoassay method of the present invention uses any one of themarkers.

An immunoassay reagent in the present invention comprises any one of theresin-metal composites.

A method for measuring analyte in the present invention is a method formeasuring analyte which can assay or quantify an analyte contained in asample.

The method for measuring analyte in the present invention uses alateral-flow chromatographic test strip which comprises a membrane and adetermination portion formed by immobilizing capturing ligandsspecifically bound with the analyte on the membrane.

Moreover, the method for measuring analyte in the present invention ischaracterized by carrying out steps including Step (I) to Step (III)hereinafter:

Step (I): Step of making the analyte contained in the sample contactwith a marked antibody formed by utilizing the resin-metal compositeaccording to any one of claims 1 to 7 to mark an antibody specificallybound with the analyte.

Step (II): Step of making the composite containing the analyte and themarked antibody formed in Step (I) contact with the capturing ligands inthe determination portion.

Step (III): Step of determining colored intensity derived from thelocalized surface plasmon resonance of the resin-metal composite.

An analyte measurement kit in the present invention is an analytemeasurement kit which uses the lateral-flow chromatographic test stripand is used to assay or quantify an analyte contained in a sample.

The analyte measurement kit in the present invention comprises: alateral-flow chromatographic test strip, comprising a membrane and adetermination portion formed by immobilizing capturing ligandsspecifically bound with the analyte on the membrane;

and an assay reagent, containing a marked antibody formed by utilizingthe resin-metal composite according to any one of claims 1 to 7 to markan antibody specifically bound with the analyte.

A lateral-flow chromatographic test strip in the present invention is alateral-flow chromatographic test strip which is used to assay orquantify an analyte contained in a sample.

The lateral-flow chromatographic test strip in the present inventioncomprises:

a membrane;

a determination portion, formed by immobilizing capturing ligandsspecifically bound with the analyte on the membrane in the spreadingdirection of a sample; and

a reaction portion, comprising an marked antibody formed by utilizingthe resin-metal composite according to any one of claims 1 to 7 to markan antibody specifically bound with the analyte further upstream thanthe determination portion.

Effects of the Present Invention

At least portions of the metal particles of the resin-metal composite ofthe present invention are three-dimensionally distributed in the surfacesection of the resin particle, moreover, portions of thethree-dimensionally distributed metal particles are partially exposedfrom the resin particle, and the rest portions are encased in the resinparticle. Therefore, the quantity of the carried metal particlesmanifesting localized surface plasmon absorption on the resin particleis high. In addition, the metal particles can hardly come off from theresin particle. Therefore, as a material with excellent durability,visual recognizability, visual determinability and assay sensitivity,the resin-metal composite of the present invention can be preferablyapplied in purposes, such as a marker for immunoassay (e.g. EIA, RIA,CLIA, FIA, LIA, PA, ICA, HA and HI), an immunoassay reagent, a solidcatalyst, a pigment, a coating, a conductive material, an electrode anda sensor component. In particular, when the resin-metal composite of thepresent invention is used as a material for immunoassay, the durabilityand visual recognizability are excellent, and high-sensitivitydetermination can be realized without requiring the addition of aspecial device or operation steps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural schematic diagram representing the section of aresin-metal composite in an embodiment of the present invention.

FIG. 2 is an explanatory diagram representing the summary of a methodfor measuring analyte using a lateral-flow chromatographic test strip inan embodiment of the present invention.

FIG. 3 is a scanning electron microscope (SEM) picture of a resin-goldcomposite obtained in embodiment 1.

FIG. 4 is a scanning electron microscope (SEM) picture of a resin-goldcomposite obtained in embodiment 2.

FIG. 5 is a scanning transmission electron microscope (STEM) picture ofthe section of the resin-gold composite obtained in embodiment 2.

FIG. 6 is a scanning electron microscope (SEM) picture of a resin-goldcomposite obtained in embodiment 3.

FIG. 7 is a scanning transmission electron microscope (STEM) picture ofthe section of the resin-gold composite obtained in embodiment 3.

FIG. 8 is a scanning electron microscope (SEM) picture of a resin-goldcomposite obtained in embodiment 10.

DESCRIPTION OF THE EMBODIMENTS

In reference to the drawings, the embodiments of the present inventionare elaborated hereinafter.

FIG. 1 is a sectional schematic diagram of a resin-metal composite in anembodiment of the present invention. A resin-metal composite 100 isprovided with resin particles 10 and metal particles 20.

The resin-metal composite 100 disperses or immobilizes the metalparticles 20 on the resin particle 10. In addition, portions of themetal particles 20 of the resin-metal composite 100 arethree-dimensionally distributed in a surface section 60 of the resinparticle 10, moreover, portions of the three-dimensionally distributedmetal particles 20 are partially exposed from the resin particle 10, andthe rest portions are encased in the resin particle 10.

Here, metal particles completely encased in the resin particle 10 (alsocalled “encased metal particles 30” hereinafter), metal particles havinga portion embedded in the resin particle 10 and a portion exposed fromthe resin particle 10 (also called “partially exposed metal particles40” hereinafter) and metal particles adsorbed on the surface of theresin particle 10 (also called “surface-adsorbed metal particles 50”hereinafter) exist among the metal particles 20. The partially exposedmetal particles 40 and the surface-adsorbed metal particles 50 areequivalent to “first particles” in the present invention, and theencased metal particles 30 are equivalent to “second particles” in thepresent invention

For example, when the resin-metal composite 100 is applied to a markerfor immunoassay or an immunoassay reagent, an antibody or an antigen isimmobilized on the partially exposed metal particles 40 or thesurface-adsorbed metal particles 50 for use. At this point, the antibodyor the antigen is immobilized on the partially exposed metal particles40 and the surface-adsorbed metal particles 50, but, on the other hand,is not immobilized on the encased metal particles 30. However, becausethe metal particles 20 including the encased metal particles 30 allmanifest localized surface plasmon absorption, not only can thepartially exposed metal particles 40 and the surface-adsorbed metalparticles 50 help to increase the visual recognizability of the markerfor immunoassay and the immunoassay reagent, but also the encased metalparticles 30 can help to increase the visual recognizability of themarker for immunoassay and the immunoassay reagent. Further, comparedwith the surface-adsorbed metal particles 50, the partially exposedmetal particles 40 and the encased metal particles 30 have large contactarea with the resin particle 10, and, besides, can hardly come off fromthe resin particle 10 due to the high adsorption force of physics suchas an anchoring effect produced by the embedded state. Therefore, thedurability and stability of the marker for immunoassay and theimmunoassay reagent using the resin-metal composite 100 becomeexcellent.

In addition, in applications except immunoassay, particularlyapplications in which the metal particles 20 play a role as a functionmanifestation portion, the same property improvement can certainly beexpected. As such applications, examples are listed as follows: a solidcatalyst, a pigment, a coating, a conductive material, an electrode anda sensor component.

The case of applying the resin-metal composite 100 in a marker forimmunoassay (called “marker” for short hereinafter) or an immunoassayreagent (called “reagent” for short hereinafter) is described as anexample hereinafter.

The whole surface of each encased metal particle 30 is covered by resinforming the resin particle 10. In addition, 5 percent to less than 100percent of the surface area of each partially exposed metal particle 40is covered by the resin forming the resin particle 10. From the point ofthe durability of each application, the lower limit is preferably 20percent or more of the surface area, more preferably 30 percent or more.In addition, more than 0 percent but less than 5 percent of the surfacearea of each surface-adsorbed metal particle 50 is covered by the resinforming the resin particle 10.

In addition, relative to the weight of the resin-metal composite 100,the quantity of the carried metal particles 20 (the total of the encasedmetal particles 30, the partially exposed metal particles 40 and thesurface-adsorbed metal particles 50) of the resin-metal composite 100 ispreferably 5 wt % to 70 wt %. If it is within the range, then the visualrecognizability, visual determinability and assay sensitivity of theresin-metal composite 100 as the marker are excellent. If the quantityof the carried metal particles 20 is less than 5 wt %, then there existsa tendency of decrease in the amount of the immobilized antibody orantigen and decrease in assay sensitivity. The quantity of the carriedmetal particles 20 is more preferably 15 wt % to 70 wt %.

In addition, preferably, 10 wt % to 90 wt % of the metal particles 20are the partially exposed metal particles 40 and the surface-adsorbedmetal particles 50. If it is within the range, then the amount of theantibody or the antigen immobilized onto the metal particles 20 can besufficiently guaranteed, so the sensitivity of it as the marker is high.More preferably, 20 wt % to 80 wt % of the metal particles 20 are thepartially exposed metal particles 40 and the surface-adsorbed metalparticles 50, and from the point of durability, more preferably, thesurface-adsorbed metal particles 50 are 20 wt % or less.

In addition, when the resin-metal composite 100 is used in immunoassay,in order to obtain excellent assay sensitivity, preferably 60 wt % to100 wt %, preferably 75 wt % to 100 wt % or more preferably 85 wt % to100 wt % of the metal particles 20 exist in the surface section 60, and,more preferably, exist within a range of 40% of particle radius in adepth direction from the surface of the resin particle 10. In addition,5 wt % to 90 wt % of the metal particles 20 existing in the surfacesection 60 are the partially exposed metal particles 40 or thesurface-adsorbed metal particles 50, sufficiently guaranteeing theamount of the antibody or the antigen immobilized onto the metalparticle 20, so the sensitivity of it as the marker becomes high and ispreferred. In other words, 10 wt % to 95 wt % of the metal particles 20existing in the surface section 60 can be the encased metal particles30.

Here, the “surface section” means a range of 50% of the particle radiusin the depth direction from the surface of the resin particle 10 withthe outermost position (i.e. the protruding ends of the partiallyexposed metal particles 40 or the surface-adsorbed metal particles 50)of the resin-metal composite 100 as a datum. In addition,“three-dimensionally distributed” means that the metal particles 20 aredistributed not only in the surface direction of the resin particle 10but also in the depth direction.

Preferably, the resin particle 10 is a polymer particle which isprovided with a substituent group capable of adsorbing metal ions in thestructure. In particular, a nitrogenous polymer particle is preferred.Nitrogen atoms in a nitrogenous polymer can easily chemically adsorb theprecursor (i.e. anionic metal ions) of a metal particle, such as gold orpalladium, which has excellent visual recognizability and can easilyimmobilize an antigen or an antibody, so it is preferred. In the presentembodiment, the metal ions adsorbed in the nitrogenous polymer arereduced, so that metal nanoparticles are formed, so portions of theproduced metal particles 20 become the encased metal particles 30 or thepartially exposed metal particles 40. In addition, because carboxylicacid and the like can adsorb cationic metal ions like acrylic acidpolymer, the precursor of a metal particle, such as silver, nickel orcopper (i.e. cationic metal ions), can be easily adsorbed, so that themetal particles 20 of silver, nickel, copper or the like can be formed,and an alloy of a metal, such as gold or palladium, can be made.

On the other hand, in the case that it is a resin particle rather thanthe nitrogenous polymer having the substituent group capable ofadsorbing metal ions in the structure, for example, in the case ofpolystyrene, the metal ions can hardly be adsorbed in resin. As aresult, the majority of the produced metal particles 20 are thesurface-adsorbed metal particles 50. As mentioned above, the contactarea between the surface-adsorbed metal particles 50 and the resinparticle 10 is small, so there exists a tendency of low bonding forcebetween the resin and the metal and great affection of the metalparticles 20 coming off from the resin particle 10.

The nitrogenous polymer is resin which has nitrogen ions on the mainchain or the side chain, for example, polyamine, polyamide, polypeptide,polyurethane, polyurea, polyimide, polyimidazole, polyoxazole,polypyrrole or polyaniline. Polyamine, such as poly-2-vinylpyridine,poly-3-vinylpyridine or poly-4-vinylpyridine, is preferred. In addition,for example, when there are nitrogen atoms on the side chain, acrylicresin, phenolic resin and epoxy resin can be widely utilized.

For example, the metal particles 20 can apply silver, nickel, copper,gold and palladium. Gold and palladium which have excellent visualrecognizability and can easily immobilize an antigen or an antibody arepreferred. These manifest absorption stemming from localized surfaceplasmon resonance, and therefore are preferred. Gold with good stabilityin storage is more preferred. These metals can be used as monomers orcomposites such as alloys. Here, for example, gold alloy means an alloywhich contains gold and metal varieties except gold, and contains 10 wt% or more of gold.

In addition, for example, the average particle size of the metalparticles 20 relaying on scanning electron microscope (SEM) observationto determine length is preferably 1 nm to 80 nm. In the case that theaverage particle size of the metal particles 20 is less than 1 nm orexceeds 80 nm, a localized surface plasmon can hardly manifest, so thereexists a tendency of decrease in sensitivity. When the metal particles20 are gold particles, the average particle size of the metal particles20 is preferably 20 nm to less than 70 nm, more preferably 22 nm to lessthan 50 nm.

In addition, for example, the average particle size of the resin-metalcomposite 100 is preferably 100 nm to 1000 nm. If the average particlesize of the resin-metal composite 100 is less than 100 nm, then, forexample, when the gold particles are used as the metal particles 20,there exists a tendency that the quantity of the carried gold particlesbecomes less, and therefore there exists a tendency that coloringbecomes weak in comparison with gold particles of the same size; and ifthe average particle size of the resin-metal composite 100 exceeds 1000nm, then there exists a tendency of easy blockage in pores of achromatographic medium, such as a membrane filter, or a tendency ofdecrease in dispersibility when the resin-metal composite 100 isprepared into a marker or a reagent. The average particle size of theresin-metal composite 100 is preferably 100 nm to less than 700 nm, morepreferably 340 nm to less than 650 nm. Here, the particle size of theresin-metal composite 100 means a value which is obtained by adding theparticle size of the resin particle 10 with the length of the protrudingportions of the partially exposed metal particles 40 or thesurface-adsorbed metal particles 50, and can be determined by a laserdiffraction/scattering method, a dynamic light scattering method or acentrifugal precipitation method.

[Preparation Method for Resin-Metal Composite]

Preparation methods for a resin-metal composite 100 are not speciallylimited. For example, solution containing metal ions is added intodispersion of resin particles 10 prepared by the emulsion polymerizationmethod, so that the metal ions are adsorbed on the resin particles 10(called “metal ion-adsorbing resin particles” hereinafter). Further, themetal ion-adsorbing resin particles are added into a reducing agentsolution, so that the metal ions are reduced to form metal particles 20,and thereby the resin-metal composite 100 is obtained.

In addition, for example, when gold particles are used as the metalparticles 20, aqueous chloroauric acid (HAuCl₄) solution can be used asthe solution containing the metal ions. In addition, a metal complex canbe used to substitute for the metal ions.

In addition, as a solvent for the solution containing the metal ions,aqueous alcohol or alcohol, such as methanol, ethanol, n-propyl alcohol,isopropyl alcohol, n-butyl alcohol, sec-butyl alcohol or tert-butylalcohol, or acid, such as hydrochloric acid, sulfuric acid or nitricacid, can be used to substitute for water.

In addition, if needed, an additive, such as various water-miscibleorganic solvents (such as water-soluble macromolecular compound (such aspolyvinyl alcohol), surfactant, alcohols, ethers (such astetrahydrofuran, diethyl ether and diisopropyl ether), polyhydricalcohols (such as alkylene glycol, polyalkylene glycol, their monoalkylether or dialkyl ether and glycerol) and ketones (such as acetone andmethyl ethyl ketone)), can be added into the solution. Such additiveaccelerates the speed of the reduction reaction of the metal ions, andis also effective in controlling the size of the produced metalparticles 20.

In addition, a well-known reducing agent can be used. Examples arelisted as follows: sodium borohydride, dimethylamine borane, citricacid, sodium hypophosphite, hydrazine hydrate, hydrazine hydrochloride,hydrazine sulfate, formaldehyde, sucrose, glucose, ascorbic acid,isoascorbic acid, sodium hypophosphite, hydroquinone and Rochelle salt.Sodium borohydride, dimethylamine borane or citric acid is preferred.

If needed, surfactant can be added into the reducing agent solution, orthe pH of the solution is regulated. The pH can be regulated bybuffering agent (such as boric acid or phosphoric acid), acid (such ashydrochloric acid or sulfuric acid) or alkali (such as sodium hydroxideor potassium hydroxide).

Further, the reduction speed of the metal ions is regulated by thetemperature of the reducing agent solution, so that the particle size ofthe formed metal particles can be controlled.

In addition, when the metal ions in the metal ion-adsorbing resinparticles are reduced to produce the metal particles 20, the metalion-adsorbing resin particles can be added into the reducing agentsolution, or the reducing agent can be added into the metalion-adsorbing resin particles, nevertheless, from the point of theeasiness of the production of encased metal particles 30 and partiallyexposed metal particles 40, the former is preferred.

In addition, in order to keep the dispersibility of the resin-metalcomposite 100 in water, for example, a dispersing agent, such as citricacid, poly-L-lysine, polyvinylpyrrolidone, polyvinylpyridine, polyvinylalcohol, disperbyk 194, disperbyk 180, disperbyk 184 (produced byBYK-Chemie Japan), can be added.

Further, pH can be regulated by a buffering agent (such as boric acid orphosphoric acid), acid (such as hydrochloric acid or sulfuric acid) oralkali (such as sodium hydroxide or potassium hydroxide), anddispersibility is kept.

Particularly, by adsorbing the antigen or antibody on the surfaces ofthe metal particles 20, the resin-metal composite 100 with theabove-mentioned composition is preferably applicable as a marker toimmunoassay, such as EIA, RIA, CLIA, FIA, LIA, PA, ICA, HA and HI. Inaddition, in particular, the resin-metal composite 100 can be preferablyused as a material for a marker for immunoassay or an immunoassayreagent which has excellent visual determinability in low-concentrationregions (high-sensitivity regions). In addition, the forms of the markerfor immunoassay or the immunoassay reagent are not specially limited,and, for example, can be used as dispersions which are formed bydispersing the resin-metal composite 100 into water or a pH-regulatedbuffer.

Methods for adsorbing the antigen or antibody onto the surface of themetal particles 20 are not specially limited, and the well-knownphysical adsorption and chemical adsorption methods can be utilized.Examples are listed as follows: physical adsorption, such as immersingthe resin-metal composite 100 into buffer containing the antigen orantibody for incubation, or chemical adsorption, such as introducing anSH group into the antigen or antibody to react with the resin-metalcomposite 100 to form an Au—SH bond. In order to make the bondingbetween the metal particles 20 and the antigen or antibody become firm,chemical adsorption is preferred.

Besides applications as the marker for immunoassay or the immunoassayreagent, the resin-metal composite 100 can be preferably used as a solidcatalyst, a pigment, a coating, a conductive material, an electrode anda sensor component.

A method for measuring analyte, a lateral-flow chromatographic teststrip and an analyte assay and quantification kit which adopt theresin-metal composite 100 as a marker are then described.

[Lateral-Flow Chromatographic Test Strip]

Firstly, in reference to FIG. 2, a lateral-flow chromatographic teststrip (test strip) in an embodiment of the present invention isdescribed. As mentioned hereinafter, the test strip 200 can bepreferably used in a method for measuring analyte in an embodiment ofthe present invention.

The test strip 200 is provided with a membrane 110. In the membrane 110,a sample addition portion 120, a determination portion 130 and aliquid-absorbing portion 140 are sequentially arranged in the spreadingdirection of a sample.

<Membrane>

A material which is used as a membrane in ordinary test strips can beapplied as the membrane 110 used in the test strip 200. For example, themembrane 110 is formed by an inert material including the followingmicroporous substances (substances not reacting with an analyte 160 andvarious ligands, etc.), the microporous substances show capillarity, andwhen added, the sample spreads. Specific examples as the membrane 110are listed as follows: fibrous or non-woven fibrous matrices and filmscontaining polyurethane, polyester, polyethylene, polyvinyl chloride,polyvinylidene fluoride, nylon and cellulose derivatives, etc., filterpaper, glass fiber filter paper, cloth and cotton. Among these,preferably, a film containing cellulose derivatives or nylon, filterpaper or glass fiber filter paper can be used, or more preferably, acellulose nitrate film, a mixed nitrocellulose ester (a mixture ofcellulose nitrate and cellulose acetate) film, a nylon film or filterpaper can be used.

In order to make operation easier, the test strip 200 is preferably asupporting body with the supporting membrane 110. For example, as thesupporting body, plastic can be used.

<Sample Addition Portion>

The test strip 200 can also be provided with the sample addition portion120 for the addition of a sample containing the analyte 160. In the teststrip 200, the sample addition portion 120 is a portion for receivingthe sample containing the analyte 160. In the spreading direction of thesample, the sample addition portion 120 can be formed on the membrane110 further upstream than the determination portion 130, or a sampleaddition pad containing a material, such as cellulose filter paper,glass fibers, polyurethane, polyacetate, cellulose acetate, nylon orcotton, can be arranged on the membrane 110 to form the sample additionportion 120.

<Determination Portion>

Capturing ligands 131 which are specifically bound with the analyte 160are immobilized in the determination portion 130. As long as thecapturing ligands 131 are specifically bound with the analyte 160, thereis no special limitation in use, for example, an antibody for theanalyte 160 can be preferably used. Even if the sample is supplied tothe test strip 200, the capturing ligands 131 are immobilized in amanner of not moving from the determination portion 130. The capturingligands 131 only need to be directly or indirectly immobilized on themembrane 110 by physical bonding or chemical bonding or adsorption.

In addition, as long as the determination portion 130 is composed like acomposite 170 containing a marked antibody 150 and the analyte 160 incontact with the capturing ligands 131 specifically bound with theanalyte 160, there is no special limitation. For example, the capturingligands 131 can be directly immobilized on the membrane 110, or thecapturing ligands 131 can be immobilized on a pad containing cellulosefilter paper, glass fibers or non-woven cloth fixed on the membrane 110.

<Liquid-Absorbing Portion>

For example, the liquid-absorbing portion 140 can be formed by a pad ofa water-absorbing material such as cellulose filter paper, non-wovencloth, cloth and cellulose acetate. The moving speed of the sample afterthe front line of spreading of the added sample arrives at theliquid-absorbing portion 140 is different according to the materials andsizes of the liquid-absorbing portion 140. Therefore, by choosing thematerial and size of the liquid-absorbing portion 140, the most suitablespeed can be set for the assay and quantification of the analyte 160.Moreover, the composition of the liquid-absorbing portion 140 isoptional, and therefore can be omitted.

If needed, the test strip 200 can also comprise any portions, such as areaction portion and a control portion.

<Reaction Portion>

Although graphical representation is omitted, in the test strip 200, thereaction portion containing the marked antibody 150 can be formed on themembrane 110. In the flowing direction of the sample, the reactionportion can be arranged further upstream than the determination portion130. Furthermore, the sample addition portion 120 in FIG. 2 can be usedas the reaction portion. When the test strip 200 has the reactionportion, if the sample containing the analyte 160 is supplied to thereaction portion or the sample addition portion 120, then, in thereaction portion, the analyte 160 contained in the sample can be incontact with the marked antibody 150. In this case, the sample is onlysupplied to the reaction portion or the sample addition portion 120, sothat the composite 170 containing the analyte 160 and the markedantibody 150 can be formed, so a so-called one-step immunochromatographycan be implemented.

As long as the reaction portion contains the marked antibody 150specifically bound with the analyte 160, there is no special limitation,and the reaction portion can be formed by directly applying the markedantibody 150 on the membrane 110. Or the reaction portion can also beformed by immobilizing a pad (conjugate pad) on the membrane 110,wherein the pad containing cellulose filter paper, glass fibers ornon-woven cloth and the marked antibody 150 is impregnated in the pad.

<Control Portion>

Although graphical representation is omitted, the control portion canalso be formed on the test strip 200 in the spreading direction of thesample by immobilizing the capturing ligands specifically bound with themarked antibody 150 on the membrane 110. By determining coloredintensity in the determination portion 130 and the control portiontogether, it can be determined that the sample supplied to the teststrip 200 arrives at the reaction portion and the determination portion130 after spreading, and examination can be normally carried out.Moreover, except using other types of capturing ligands specificallybound with the marked antibody 150 to replace the capturing ligands 131,the control portion can be made in the same way as the determinationportion 130, and can adopt the same composition.

[Method for Measuring Analyte]

A method for measuring the analyte 160 using the test strip 200 in anembodiment of the present invention is then described.

The method for measuring the analyte 160 in the present embodiment is amethod for measuring the analyte 160 which can assay or quantify theanalyte 160 contained in the sample. The method for measuring theanalyte 160 in the present invention can use the test strip 200comprising the membrane 110 and the determination portion 130 formed byimmobilizing the capturing ligands 131 specifically bound with theanalyte 160 on the membrane 110, and comprises Step (I) to Step (III)below:

Step (I): Step of making the analyte 160 contained in the sample contactwith the marked antibody 150 formed by utilizing the resin-metalcomposite 100 having the structure formed by immobilizing the pluralityof metal particles 20 on the resin particle 10 to mark an antibodyspecifically bound with the analyte 160.

Step (II): Step of making the composite containing the analyte 160 andthe marked antibody 150 formed in Step (I) contact with the capturingligands 131 in the determination portion 130.

Step (III): Step of determining colored intensity derived from thelocalized surface plasmon resonance of the resin-metal composite 100.

Step (I):

Step (I) is a step of making the analyte 160 contained in the samplecontact with the marked antibody 150. As long as the composite 170containing the analyte 160 and the marked antibody 150 is formed, thereis no special limitation in the form of contact.

For example, the sample can be supplied to the sample addition portion120 or reaction portion (graphical representation omitted) of the teststrip 200 and the analyte 160 is made contact with the marked antibody150 in the reaction portion, or, before the sample is supplied to thetest strip 200, the analyte 160 in the sample can be made contact withthe marked antibody 150.

The composite 170 formed in Step (I) is moved after spreading on thetest strip 200, and arrives at the determination portion 130.

Step (II):

Step (II) is a step of making the composite 170 containing the analyte160 and the marked antibody 150 formed in Step (I) contact with thecapturing ligands 131 in the determination portion 130 of the test strip200. If the composite 170 is made contact with the capturing ligands131, then the capturing ligands 131 are specifically bound with theanalyte 160 of the composite 170. As a result, the composite 170 iscaptured in the determination portion 130.

Furthermore, because the capturing ligands 131 are not specificallybound with the marked antibody 150, when the marked antibody 150 notbound with the analyte 160 arrives at the determination portion 130, themarked antibody 150 not bound with the analyte 160 passes through thedetermination portion 130. Here, when the control portion (graphicalrepresentation omitted) on which other capturing ligands specificallybound with the marked antibody 150 are immobilized is formed in the teststrip 200, the marked antibody 150 which has passed through thedetermination portion 130 continues to spread, and is bound with theother capturing ligands in the control portion. As a result, the markedantibody 150 which does not form the composite 170 along with theanalyte 160 is captured in the control portion.

After Step (II), if needed, before Step (III), for example, a cleaningstep of cleaning the test strip 200 by utilizing buffer commonly used inbiochemical examination, such as water, normal saline or phosphatebuffer, can be implemented. By means of the cleaning step, the markedantibody 150 (marked antibody 150 not bound with the analyte 160 so asnot to form the composite 170) which is not captured in thedetermination portion 130 or the determination portion 130 and thecontrol portion can be removed.

By implementing the cleaning step, when coloration caused by thelocalized surface plasmon resonance of the resin-metal composite 100 inthe determination portion 130 or the determination portion 130 and thecontrol portion is determined in Step (III), the colored intensity ofthe background can be decreased, the signal/background ratio can beincreased, and assay sensitivity or quantifiability can be furtherincreased.

Step (III):

Step (III) is a step of determining colored intensity derived from thelocalized surface plasmon resonance of the resin-metal composite 100.After Step (II) or the cleaning step, if needed, is implemented, in thetest strip 200, colored intensity derived from the localized surfaceplasmon resonance of the resin-metal composite 100 is determined.

Moreover, when the control portion is formed in the test strip 200, bymeans of Step (II), in the control portion, the marked antibody 150 iscaptured by the other capturing ligands to form the composite.Therefore, in Step (III), in the test strip 200, not only can colorationcaused by localized surface plasmon resonance be generated in thedetermination portion 130, but also coloration caused by localizedsurface plasmon resonance be generated in the control portion. Thus, bydetermining colored intensity in the determination portion 130 and thecontrol portion together, whether the sample supplied to the test strip200 arrives at the reaction portion and the determination portion 130after spreading normally can be determined.

<Sample and Analyte>

As long as the sample in the method for measuring analyte in the presentembodiment contains a substance capable of becoming an antigen, such asprotein, as the analyte 160, there is no special limitation. Examplesare listed as follows: an organism sample (i.e. whole blood, serum,plasma, urine, saliva, phlegm, rhinal swab fluid or pharyngeal swabfluid, spinal fluid, amniotic fluid, nipple secretion, tear, sweat,extract coming from skin, extract coming from tissues or cells andfeces, etc.) containing the target analyte 160 or extract of food. Ifneeded, in order to easily cause the specific binding reaction betweenboth the marked antibody 150 and the capturing ligands 131 and theanalyte 160, before Step (I), the analyte 160 contained in the sample ispretreated. Here, as pretreatment, chemical treatment utilizing variouschemicals (such as acid, alkali and surfactant) or physical treatmentutilizing heating, agitation and ultrasonic waves can be adopted. Inparticular, when the analyte 160 is a substance normally not exposed tothe surface, such as an influenza virus NP antigen, surfactant ispreferably utilized for treatment. As the surfactant for this purpose,non-ionic surfactant can be used in consideration of specific bindingreaction, such as binding reactivity between ligands forantigen-antibody reaction and the analyte 160.

In addition, the sample can be properly diluted by a solvent (such aswater, normal saline or buffer) or a water-miscible organic solventnormally used in immunological analysis.

As the analyte 160, examples are listed as follows: proteins such astumor pointers, signal transmission substances and hormone (includingpolypeptide, oligopeptide and the like), nucleic acids (includingsingle-stranded or double-stranded deoxyribonucleic acid (DNA),ribonucleic acid (RNA) and polynucleotide, oligonucleotide, peptidenucleic acid (PNA) and the like) or substances with nucleic acid,saccharides (including oligosaccharide, polysaccharides, carbohydratechain and the like) or substances with carbohydrate chains, and othermolecules such as lipid. As long as it can be specifically bound withthe marked antibody 150 and the capturing ligands 131, there is nospecial limitation. Examples are listed as follows: carcino-embryonicantigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9,α-fetoprotein (AFP), immunosuppressive acidic protein (IAP), CA15-3,CA125, estrogen receptor, progesterone receptor, fecal occult blood,troponin 1, troponin T, CK-MB, CRP, human chorionic gonadotrophin (HCG),luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilisantibody, influenza virus human hemoglobin, chlamydia antigen, group A βhemolytic streptococcus antigen, HBs antibody, HBs antigen, rotavirus,adenovirus, albumin and glycated albumin. Among these, antigen which canbe dissolved by non-ionic surfactant is preferred, and antigen which isformed from self-aggregate like the nucleoprotein of virus is morepreferred.

<Marked Antibody>

The marked antibody 150 is used in Step (I) to contact with the analyte160 contained in the sample, so that the composite 170 containing theanalyte 160 and the marked antibody 150 is formed. The marked antibody150 is formed by utilizing the resin-metal composite 100 having thestructure formed by immobilizing the plurality of metal particles 20 onthe resin particle 10 to mark an antibody specifically bound with theanalyte 160. Here, the so-called “marking” means directly or indirectlyimmobilizing the resin-metal composite 100 onto the antibody by chemicalbinding or physical binding or adsorption on the basis of the degree ofthe resin-metal composite 100 not coming off from the marked antibody150 in Step (I) to Step (III). For example, the marked antibody 150 canbe formed by directly binding the antibody with the resin-metalcomposite 100, or can be formed by binding the antibody with theresin-metal composite 100 through any linker molecules or respectivelyimmobilizing them on insoluble particles.

In addition, in the present embodiment, there is no special limitationon “antibody”, for example, besides polyclonal antibodies, monoclonalantibodies and antibodies obtained by genetic recombination, antibodyfragments (such as H chain, L chain, Fab and F(ab′)₂) capable of beingbound with antigens can be used. In addition, immune globulin can be anyone of IgG, IgM, IgA, IgE and IgD. Animal species producing antibodiescan be the human being and animals (e.g. mouse, rat, rabbit, goat andhorse) except the human being. Specific examples as the antibody arelisted as follows: anti-PSA antibody, anti-AFP antibody, anti-CEAantibody, anti-adenovirus antibody, anti-influenza virus antibody,anti-HCV antibody, anti-IgG antibody and anti-human IgE antibody.

<Preferred Preparation Method for Marked Antibody>

A preferred preparation method for the marked antibody 150 is thendescribed. The preparation of the marked antibody 150 can at leastcomprise Step A below:

Step A: Step of binding the resin-metal composite 100 with the antibodyby mixing under a first pH condition to obtain the marked antibody 150,and preferably, Step B is further included;

Step B: Step of treating the marked antibody 150 under a second pHcondition.

[Step A]

In Step A, under the first pH condition, the resin-metal composite 100and the antibody are mixed, so that the marked antibody 150 is obtained.Preferably, in Step A, the solid resin-metal composite 100 contacts withthe antibody in the state of being dispersed in the liquid phase. Thefirst pH condition is different according to the metal varieties of themetal particles 20 in the resin-metal composite 100.

When the metal particles 20 of the resin-metal composite 100 are goldparticles (including gold alloy particles; the same is truehereinafter), from the point of making the resin-metal composite 100uniformly contact with the antibody under the state of maintaining thedispersion of the resin-metal composite 100 and the activity of theantibody in order to be bound with the antibody, the first pH conditionis preferably a condition of pH within a range from 2 to 7, and further,an acidic condition is more preferred, for example, pH is within a rangefrom 2.5 to 5.5. When the metal particles 20 are gold particles, if thecondition in binding the resin-metal composite 100 with the antibody ispH less than 2, then there will exist a situation that the antibody isdeteriorated and inactivated due to strong acidity; and if pH exceeds 7,then the resin-metal composite 100 will be aggregated and hardlydispersed when mixed with the antibody. However, when the antibody isnot inactivated due to strong acidity, even if pH is less than 2,treatment can still be carried out.

In addition, when the metal particles 20 of the resin-metal composite100 are particles rather than gold particles, e.g. palladium particlesor their alloys, from the point of making the resin-metal composite 100uniformly contact with the antibody under the state of maintaining thedispersion of the resin-metal composite 100 and the activity of theantibody in order to be bound with the antibody, the first pH conditionis preferably a condition of pH within a range from 2 to 10, and morepreferably, for example, pH is within a range from 5 to 9. When themetal particles 20 are particles rather than gold particles, if thecondition in binding the resin-metal composite 100 with the antibody ispH less than 2, then there will exist a situation that the antibody isdeteriorated and inactivated due to strong acidity; and if pH exceeds10, then the resin-metal composite 100 will be aggregated and hardlydispersed when mixed with the antibody. However, when the antibody isnot inactivated due to strong acidity, even if pH is less than 2,treatment can still be carried out.

Preferably, Step A is carried out in binding buffer regulated to thefirst pH condition. For example, a specified amount of resin-metalcomposite 100 is mixed in the binding buffer regulated to the pH, and issufficiently mixed. For example, as the binding buffer, boric acidsolution regulated to specified concentration can be used. For example,the pH of the binding buffer can be regulated by using hydrochloricacid, sodium hydroxide, etc.

Afterwards, a specified amount of antibody is added into the obtainedmixture, and is sufficiently agitated and mixed, and thereby a markedantibody-containing solution can be obtained. For example, only themarked antibody 150 as solid part is separated out from the markedantibody-containing solution obtained in this way by a solid-liquidseparation method, such as centrifugal separation.

[Step B]

In Step B, under the second pH condition, the marked antibody 150obtained in Step A is treated in order to carry out blockage ofinhibiting the non-specific adsorption of the marked antibody 150. Inthis case, under the second pH condition, the marked antibody 150 whichis separated out by the solid-liquid separation method is dispersed inthe liquid phase. The condition of blockage is different according tothe metal varieties of the metal particles 20 in the resin-metalcomposite 100.

When the metal particles 20 of the resin-metal composite 100 are goldparticles, from the point of keeping the activity of the antibody andinhibiting the aggregation of the marked antibody 150, for example, thesecond pH condition is preferably pH within a range from 2 to 9, andfurther, from the point of inhibiting the non-specific adsorption of themarked antibody 150, an acidic condition is more preferred, for example,pH is within a range from 2 to 6. If the condition of blockage is pHless than 2, then there will exist a situation that the antibody isdeteriorated and inactivated due to strong acidity; and if pH exceeds 9,then the marked antibody 150 will be aggregated and hardly dispersed.

In addition, when the metal particles 20 of the resin-metal composite100 are particles rather than gold particles, from the point of keepingthe activity of the antibody and inhibiting the aggregation of themarked antibody 150, for example, the second pH condition is preferablypH within a range from 2 to 10, and from the point of inhibiting thenon-specific adsorption of the marked antibody 150, more preferably, pHis within a range from 5 to 9. If the condition of blockage is pH lessthan 2, then there will exist a situation that the antibody isdeteriorated and inactivated due to strong acidity; and if pH exceeds10, then the marked antibody 150 will be aggregated and hardlydispersed.

Preferably, Step B is carried out by using blocking buffer regulated tothe second pH condition. For example, the blocking buffer regulated tothe pH is added into a specified amount of marked antibody 150, and themarked antibody 150 is uniformly dispersed into the blocking buffer. Forexample, preferably, as the blocking buffer, a solution of protein notbound with an assayed substance is used. As the protein capable of beingused in the blocking buffer, examples are listed as follows: bovineserum albumin, ovalbumin, casein and gelatin. More specifically,preferably, a bovine serum albumin solution regulated to specifiedconcentration is used. For example, the pH of the blocking buffer can beregulated by using hydrochloric acid, sodium hydroxide, etc.

Preferably, the marked antibody 150 can be dispersed by using adispersion method, such as ultrasonic treatment. Dispersion in which themarked antibody 150 is uniformly dispersed is obtained in this way.

Dispersion of the marked antibody 150 can be obtained in this way. Forexample, only the marked antibody 150 as solid part is separated outfrom the dispersion by a solid-liquid separation method, such ascentrifugal separation. In addition, if needed, cleaning treatment,storage treatment and the like can be implemented. Cleaning treatmentand storage treatment are described hereinafter.

(Cleaning Treatment)

Cleaning treatment adds cleaning buffer into the marked antibody 150separated out by the solid-liquid separation method, and uniformlydisperses the marked antibody 150 into the cleaning buffer. For example,preferably, a dispersion method, such as ultrasonic treatment, is usedfor dispersion. There is no limitation on the cleaning buffer, forexample, Tris(hydroxymethyl)aminomethane buffer, glycinamide buffer orarginine buffer of specified concentration regulated to pH within arange from 8 to 9 can be used. For example, the pH of the cleaningbuffer can be regulated by using hydrochloric acid, sodium hydroxide,etc. If needed, the cleaning treatment of the marked antibody 150 can berepeated multiple times.

(Storage Treatment)

During storage treatment, storage buffer is added into the markedantibody 150 separated out by the solid-liquid separation method, andthe marked antibody 150 is uniformly dispersed into the storage buffer.For example, preferably, a dispersion method, such as ultrasonictreatment, is used for dispersion. For example, as the storage buffer, asolution which is prepared by adding anticoagulant and/or stabilizer ofspecified concentration into the cleaning buffer can be used. Forexample, as the anticoagulant, saccharides represented by sucrose,maltose, lactose and trehalose or polyhydric alcohols represented byglycerol and polyvinyl alcohol can be used. There is no limitation onthe stabilizer, for example, protein, such as bovine serum albumin,ovalbumin, casein or gelatin, can be used. The storage treatment of themarked antibody 150 can be carried out in this way.

In each above-mentioned step, if needed, surfactant or preservative,such as sodium azide or paraben, can be used,

[Analyte Assay and Quantification Kit]

The analyte measurement kit in an embodiment of the present invention isa kit which is used to assay or quantify the analyte 160 contained inthe sample by using the lateral-flow chromatographic test strip 200according to the method for measuring analyte in the present embodiment.

The kit of the present embodiment comprises:

the lateral-flow chromatographic test strip 200, comprising the membrane110 and the determination portion 130 formed by immobilizing thecapturing ligands specifically bound with the analyte 160 on themembrane 110; and

an assay reagent, comprising the marked antibody 150 formed by utilizingthe resin-metal composite 100 having the structure formed byimmobilizing the plurality of metal particles 20 on the resin particle10 to mark an antibody specifically bound with the analyte 160. Ifneeded, the kit of the present embodiment can further comprise othercomponents.

When the kit of the present invention is in use, after Step (I) isimplemented by making the analyte 160 in the sample contact with themarked antibody 150 in the assay reagent, the sample is supplied to thereaction portion or sample addition portion 120 of the test strip 200,and Step (II) and Step (III) are then sequentially implemented. Or theassay reagent can be applied further upstream than the determinationportion 130 of the test strip 200, it is suitable to add the sample onthe formed reaction portion or place further upstream than the reactionportion (e.g. the sample addition portion 120) after the reactionportion is formed by drying, and Step (I) to Step (III) are thensequentially implemented.

Embodiment

The present invention is then described in detail in reference toembodiments, however, the present invention is not limited by theseembodiments. In the following embodiments and comparative examples,various determinations and evaluations are based on the following way,unless otherwise indicated.

<Determination of Absorbance of Resin-Metal Composite>

With regard to the absorbance of the resin-metal composite, resin-metalcomposite dispersion (dispersion medium: water) prepared to 0.01 wt % isadded into an optical whiteboard glass unit (the optical path length is10 mm), and an instant multi-channel photometric system (produced byOtsuka Electronics Co., Ltd., MCPD-3700) is used to determine absorbanceof 570 nm in the case of gold. In the case of gold, absorbance in 570 nmwhich is 0.9 or more is set as ◯(Good), absorbance in 570 nm which is0.5 to less than 0.9 is set as Δ (Acceptable), and absorbance in 570 nmwhich is less than 0.5 is set as x (Unacceptable).

<Solid Component Concentration Determination and Carried Metal AmountDetermination>

1 g of dispersion before concentration regulation is added into amagnetic crucible, and is treated by heat under 70° C. for 3 hours.Weights before and after heat treatment are determined, and solidcomponent concentration is worked out by the following formula.

solid component concentration (wt %)=[weight after drying (g)/weightbefore drying (g)]×100

In addition, under 500° C., the sample treated by heat is furthertreated by heat for 5 hours, weights before and after heat treatment aredetermined, and the carried metal amount is worked out by the followingformula.

carried metal amount (wt %)=[weight after heat treatment under 500° C.(g)/weight before heat treatment under 500° C. (g)]×100

<Determination of Average Particle Size of Resin-metal Composite>

A disc centrifuge type particle size distribution determination device(CPS Disc Centrifuge DC24000 UHR, produced by CPS instruments, Inc.) isused for determination. Determination is carried out in the state of theresin-metal composite dispersed in water.

<Evaluation Utilizing Immunochromatography>

Determination utilizing immunochromatography shown hereinafter iscarried out by using resin-metal composite-marked antibody dispersionprepared in each embodiment, and the performance of the resin-metalcomposite dispersion is evaluated.

(Evaluation Method)

Evaluation is carried out by using a monochrome screen for influenza Aevaluation (produced by Adtec company), and coloration levels after 5minutes, 10 minutes and 15 minutes are compared. In performanceevaluation, two-fold dilution (1 to 1024 folds) of an influenza Apositive control (APC) (the concentration of virus before APC dilutionis 5000 FFU/ml) is used as an antigen.

(Evaluation Procedure)

3 μl of resin-metal composite-marked antibody dispersion is added intoeach well of a 96-well plate, and 100 μl of two-fold dilution (1 to 1024folds) of the APC and 100 μl of negative control are mixed. Afterwards,50 μl is added into the monochrome screen for influenza A evaluation,and coloration levels after 5 minutes, 10 minutes and 15 minutes areevaluated. A color sample for colloidal gold determination (produced byAdtec company) is used to determine the coloration levels.

<Determination of Average Particle Size of Metal Particles>

The determination of the average particle size of the metal particlesdetermines the surface mean diameter of the metal particles according toan image of a substrate produced by dripping the resin-metal compositedispersion into metallic meshes with carbon supporting membranesobserved by a field emission scanning electron microscope (FE-SEM;produced by Hitachi High-Technologies, SU-9000).

Comparative Example 1

<Immunochromatography Evaluation>

100 μg of influenza antibody is mixed into 1 ml (0.1 wt %) of coloringlatex (produced by Merck Millipore company, coloring Estapor functionalparticle, K1030, average particle size: 392 nm, absorbance in 570 nm:0.83, absorbance in 400 nm: 1.11), and is agitated under roomtemperature for about 3 hours, so that the coloring latex and theantibody are bound. Bovine serum albumin solution is added until finalconcentration is changed into 1%, and is agitated under room temperaturefor 2 hours to block the coloring latex. A coloring latex-markedantibody is prepared by recovery after 5 minutes of centrifugalseparation at 12000 rpm under 4° C. and suspension in buffer containing0.2% of bovine serum albumin.

The prepared coloring latex-marked antibody is used to carry outdetermination utilizing immunochromatography shown below, and theperformance of the coloring latex is evaluated.

(Evaluation Method)

Evaluation is carried out by using the monochrome screen for influenza Aevaluation (produced by Adtec company), and coloration levels after 5minutes, 10 minutes and 15 minutes are compared. In performanceevaluation, two-fold dilution (1 to 1024 folds) of an influenza Apositive control (APC) (the concentration of virus before APC dilutionis 5000 FFU/ml) is used as an antigen.

(Evaluation Procedure) 3 μl of coloring latex-marked antibody is addedinto each well of the 96-well plate, and 100 μl of two-fold dilution (1to 1024 folds) of the APC and 100 μl of negative control are mixed.Afterwards, 50 μl is added into the monochrome screen for influenza

A evaluation, and coloration levels after 5 minutes, 10 minutes and 15minutes are evaluated. The result is represented below.

TABLE 1 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Coloring latex Antigendilution APC × APC × APC × APC × APC × APC × APC × APC × APC × APC × APC× Negative 1 2 4 8 16 32 64 128 256 512 1024 control Coloration After 53.5 2.0 1.0 0.5 0.1 0 0 0 0 0 0 0 level minutes After 10 4.0 2.5 1.5 0.50.1 0.1 0 0 0 0 0 0 minutes After 15 4.5 3.5 2.5 1.0 0.5 0.1 0.1 0 0 0 00 minutes

According to Table 1, it can be determined that the coloringlatex-marked antibody shows good coloration for the antigen diluted 16folds.

Comparative Example 2

<Synthesis of Colloidal Gold>

250 ml of 1 mM aqueous chloroauric acid solution is added into a 500 mlthree-neck round-bottomed flask, a heating reflux device is used to boilthe aqueous chloroauric acid solution as the aqueous chloroauric acidsolution is violently agitated, 25 ml of 38.8 mM aqueous sodium citratesolution is added after boiling, and whether the solution is changedfrom light yellow to deep red is determined. After continuing to beheated for 10 minutes as the solution is agitated, the solution isagitated under room temperature for about 30 minutes and left alone tobe cooled. A membrane filter, the pore diameter of which is is used tofilter the solution, and the solution is transferred into a conicalflask and stored in the shade. The average particle size of the preparedparticles is 12.3 nm. In addition, the result of absorbance determinedaccording to the method is 1.32.

<Immunochromatography Evaluation>

100 μg of influenza antibody is mixed into 1 ml of obtained colloidalgold (OD=10), and is agitated under room temperature for about 3 hours,so that the colloidal gold is bound with the antibody. Bovine serumalbumin solution is added until final concentration is changed into 1%,and is agitated under room temperature for 2 hours to block the surfaceof the colloidal gold. A colloidal gold-marked antibody is prepared byrecovery after 5 minutes of centrifugal separation at 12000 rpm under 4°C. and suspension in buffer containing 0.2% of bovine serum albumin.

The prepared colloidal gold-marked antibody is used to carry outdetermination utilizing immunochromatography shown below, and theperformance of the colloidal gold is evaluated.

(Evaluation Method)

Evaluation is carried out in the same way as comparative example 1.

(Evaluation Procedure)

3 μl of colloidal gold-marked antibody is added into each well of the96-well plate, and 100 μl of two-fold dilution (1 to 1024 folds) of theAPC and 100 μl of negative control are mixed. Afterwards, 50 μl is addedinto the monochrome screen for influenza A evaluation, and colorationlevels after 5 minutes, 10 minutes and 15 minutes are evaluated. Theresult is represented below.

TABLE 2 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Colloidal gold Antigendilution APC × APC × APC × APC × APC × APC × APC × APC × APC × APC × APC× Negative 1 2 4 8 16 32 64 128 256 512 1024 control Coloration After 54.0 3.0 2.0 1.5 0.5 0.1 0 0 0 0 0 0 level minutes After 10 4.5 3.5 2.52.0 1.0 0.5 0.1 0 0 0 0 0 minutes After 15 5.0 4.5 3.5 3.0 2.0 1.0 0.10.1 0 0 0 0 minutes

According to Table 2, it can be determined that the colloidalgold-marked antibody shows good coloration for the antigen diluted 32folds.

Embodiment 1

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 2-vinylpyridine (2-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 2 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 420 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 60 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % of gold ion-adsorbing resin particle dispersion (42.4 ml) isthen added into 1580 ml of pure water, 528 mM aqueous dimethylamineborane solution (10 ml) is dripped for 2 minutes as agitation isperformed at 160 rpm under 20° C., agitation is then performed underroom temperature for 2 hours, and thereby a resin-gold composite, theaverage particle size of which is 438 nm, is obtained. The resin-goldcomposite is precipitated by centrifugal separation (3100 rpm, 60minutes), supernatant is removed, the resin-gold composite is dispersedinto pure water again, this operation is repeated three times, refiningand concentration regulation are performed by dialysis, and thereby 1 wt% resin-gold composite dispersion is obtained. The result of absorbanceof the prepared resin-gold composite determined according to the methodis 0.98. In addition, the average particle size of the formed goldparticles is 25.0 nm, and the amount of carried gold is 54.7 wt %. Thescanning electron microscope (SEM) picture of the surface of theobtained resin-gold composite is shown in FIG. 3. In the resin-goldcomposite, the gold particles include encased gold particles completelyencased in the resin particle, partially exposed gold particles having aportion embedded in the resin particle and a portion exposed from theresin particle, and surface-adsorbed gold particles absorbed on thesurface of the resin particle, and at least portions of the goldparticles are three-dimensionally distributed on the surface section ofthe resin particle.

<Immunochromatography Evaluation>

100 μg of influenza antibody is mixed into 1 ml of obtained resin-goldcomposite dispersion (0.1 wt %), and agitation is performed under roomtemperature for about 3 hours, so that the resin-gold composite is boundwith the antibody. Bovine serum albumin solution is added until finalconcentration is changed into 1%, and agitation is performed under roomtemperature for 2 hours to block the surface of the resin-goldcomposite. Resin-gold composite-marked antibody dispersion is preparedby recovery after 5 minutes of centrifugal separation at 12000 rpm under4° C. and suspension in buffer containing 0.2% of bovine serum albumin.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 3 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 4.5 4.0 3.5 3.0 2.01.5 0.5 0.5 0.1 0 0 0 level minutes After 10 5.5 5.0 4.5 3.5 3.0 2.0 1.50.5 0.5 0.1 0 0 minutes After 15 6.5 6.0 5.0 4.5 3.5 2.5 2.0 1.0 0.5 0.10 0 minutes

According to Table 3, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 2

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (2.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 2-vinylpyridine (2-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 2 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 380 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 60 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 20° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 399 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is0.96. In addition, the average particle size of the formed goldparticles is 25.0 nm, and the amount of carried gold is 53.2 wt %. Thescanning electron microscope (SEM) picture of the surface of theobtained resin-gold composite is shown in FIG. 4, and the scanningtransmission electron microscope (STEM) picture of its section is shownin FIG. 5. In the resin-gold composite, the gold particles includeencased gold particles completely encased in the resin particle,partially exposed gold particles having a portion embedded in the resinparticle and a portion exposed from the resin particle, andsurface-adsorbed gold particles absorbed on the surface of the resinparticle, and at least portions of the gold particles arethree-dimensionally distributed on the surface section of the resinparticle. Moreover, 100% of the gold particles exist on the surfacesection.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 4 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 4.5 4.0 3.5 3.0 2.51.5 1.0 0.5 0.1 0 0 0 level minutes After 10 5.5 5.0 4.5 4.0 3.5 2.5 1.50.5 0.5 0.1 0 0 minutes After 15 6.5 6.0 5.0 4.5 4.0 3.0 1.5 1.0 0.5 0.10 0 minutes

According to Table 4, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 3

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (3.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 2-vinylpyridine (2-VP, 49.50 g) anddivinyl benzene (DVB, 0.50 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.250 g)dissolved in 18.00 g of pure water is dripped for 2 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 370 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 60 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 20° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 393 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is0.92. In addition, the average particle size of the formed goldparticles is 14.9 nm, and the amount of carried gold is 55.8 wt %. Thescanning electron microscope (SEM) picture of the surface of theobtained resin-gold composite is shown in FIG. 6, and the scanningtransmission electron microscope (STEM) picture of its section is shownin FIG. 7. In the resin-gold composite, the gold particles includeencased gold particles completely encased in the resin particle,partially exposed gold particles having a portion embedded in the resinparticle and a portion exposed from the resin particle, andsurface-adsorbed gold particles absorbed on the surface of the resinparticle, and at least portions of the gold particles arethree-dimensionally distributed on the surface section of the resinparticle. Moreover, 71% of the gold particles exist on the surfacesection.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 5 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 4.0 3.0 1.0 0.5 0.10 0 0 0 0 0 0 level minutes After 10 4.5 3.5 2.0 1.5 1.0 0.5 0.1 0 0 0 00 minutes After 15 5.0 4.0 3.0 2.5 2.0 1.0 0.5 0.1 0 0 0 0 minutes

According to Table 5, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted64 folds.

If the section image of FIG. 5 of embodiment 2 and the section image ofFIG. 7 of embodiment 3 are compared, then the assay sensitivity ofimmunochromatography of embodiment 2 in which 75% to 100% of the goldparticles exist within a range of 50% of particle radius in a depthdirection from the surface of the resin particle is more excellent.

Embodiment 4

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (5.00 g) andpolyethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 389.5 g of pure water, 2-vinylpyridine (2-VP, 48.00 g)and divinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 50.00 g of pure water is dripped for 2 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 200 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 60 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwardsconcentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, the mixed solution of 528 mM aqueousdimethylamine borane solution (10 ml) and 528 mM aqueous boric acidsolution (10 ml) is dripped for 4 minutes as agitation is performed at160 rpm under 20° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 250 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is1.69. In addition, the average particle size of the formed goldparticles is 75.0 nm, and the amount of carried gold is 52.3 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 6 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 5.0 3.0 2.0 1.0 0.10 0 0 0 0 0 0 level minutes After 10 5.5 4.0 3.0 1.0 0.5 0.1 0 0 0 0 0 0minutes After 15 5.5 4.5 4.0 3.5 1.0 0.1 0 0 0 0 0 0 minutes

According to Table 6, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted16 folds.

Embodiment 5

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 2-vinylpyridine (2-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 0.5 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 500 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 40 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 20° C., agitation is then performed under room temperaturefor 2 hours, and thereby the resin-gold composite, the average particlesize of which is 510 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is1.01. In addition, the average particle size of the formed goldparticles is 46.3 nm, and the amount of carried gold is 54.2 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 7 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × 1 APC× 2 APC × 4 APC × 8 APC × 16 APC × 32 APC × APC × APC × APC × APC ×Negative 64 128 256 512 1024 control Color- After 5 5.0 4.5 3.5 3.0 2.51.5 1.0 0.5 0.1 0.1 0 0 ation minutes level After 10 6.0 5.5 4.5 4.0 3.52.5 2.0 1.0 0.5 0.1 0 0 minutes After 15 7.0 6.5 5.5 4.5 4.0 3.0 2.0 1.00.5 0.1 0.1 0 minutes

According to Table 7, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 6

After Aliquat 336 [produced by Aldrich company] (0.50 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 2-vinylpyridine (2-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 0.5 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 613 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 40 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 3° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 625 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is0.98. In addition, the average particle size of the formed goldparticles is 25.0 nm, and the amount of carried gold is 55.3 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 8 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 5.0 4.5 3.5 3.0 2.51.5 1.0 0.5 0.5 0.1 0 0 level minutes After 10 6.0 5.5 4.5 4.0 3.5 2.52.0 1.0 0.5 0.1 0.1 0 minutes After 15 7.0 6.5 5.5 4.5 4.0 3.0 2.0 1.00.5 0.1 0.1 0 minutes

According to Table 8, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 7

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 4-vinylpyridine (4-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 2 minutes, agitationis performed at 150 rpm under 60° C. for 3.5 hours, and thereby resinparticles, the average particle size of which is 438 nm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 45 minutes), supernatant is removed, the resin particles aredispersed into pure water again, and after this operation is performedthree times, impurities are removed by dialysis. Afterwards,concentration is regulated, and thereby 10 wt % resin particledispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 3° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 448 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is0.99. In addition, the average particle size of the formed goldparticles is 24.0 nm, and the amount of carried gold is 55.7 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 9 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 5.0 4.5 3.5 3.0 2.51.5 1.0 0.5 0.1 0 0 0 level minutes After 10 6.0 5.5 4.5 4.0 3.5 2.5 2.01.0 0.5 0.1 0 0 minutes After 15 7.0 6.5 5.5 4.5 4.0 3.0 2.0 1.0 0.5 0.10.1 0 minutes

According to Table 9, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 8

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 10.00 g) aredissolved into 300 g of pure water, 3-vinylpyridine (3-VP, 48.00 g) anddivinyl benzene (DVB, 2.00 g) are added, and under nitrogen flow,agitation is performed at 150 rpm under 30° C. for 50 minutes, and isthen performed under 60° C. for 30 minutes. After agitation,2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA, 0.500 g)dissolved in 18.00 g of pure water is dripped for 2 minutes, agitationat 150 rpm under 60° C. for 3.5 hours, and thereby resin particles, theaverage particle size of which is 429 nm, are obtained. The resinparticles are precipitated by centrifugal separation (9000 rpm, 45minutes), supernatant is removed, the resin particles are dispersed intopure water again, and after this operation is performed three times,impurities are removed by dialysis. Afterwards, concentration isregulated, and thereby 10 wt % resin particle dispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 3° C., agitation is then performed under room temperaturefor 2 hours, and thereby a resin-gold composite, the average particlesize of which is 436 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is1.03. In addition, the average particle size of the formed goldparticles is 24.3 nm, and the amount of carried gold is 55.5 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 10 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC× APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 5.0 4.5 3.5 3.0 2.51.5 1.0 0.5 0.1 0 0 0 level minutes After 10 6.0 5.5 4.5 4.0 3.5 2.2 2.00.5 0.1 0 0 0 minutes After 15 7.0 6.5 5.5 4.5 4.0 3.0 2.0 1.0 0.5 0.1 00 minutes

According to Table 10, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

Embodiment 9

After 2-(Diisopropylamino)ethyl methacrylate (DPA, 10.3 g),poly(propylene glycol)diacrylate (0.2 g) and poly(ethylene glycol)methyl ether methacrylate (PEGMA, 2.0 g) are dissolved into 85 g of purewater, and under nitrogen flow, agitation is performed at 150 rpm under30° C. for 50 minutes, and is then performed under 70° C. for 30minutes. After agitation, ammonium persulphate (APS, 0.10 g) dissolvedin 2.00 g of pure water is dripped for 2 minutes, and is agitated at 150rpm under 70° C. for 3.5 hours, and thereby resin particles, the averageparticle size of which is 338 nm, are obtained. The resin particles areprecipitated by centrifugal separation (9000 rpm, 45 minutes),supernatant is removed, the resin particles are dispersed into purewater again, and after this operation is performed three times,impurities are removed by dialysis. Afterwards, concentration isregulated, and thereby 10 wt % resin particle dispersion is obtained.

After 1233 ml of pure water is added into the resin beads (50 ml), 30 mMaqueous chloroauric acid solution (100 ml) is added, and the resin beadsare kept under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3100 rpm, 30minutes), supernatant is removed, this operation is repeated threetimes, and thereby redundant chloroauric acid is removed. Afterwards,concentration is regulated, and thereby 2.5 wt % gold ion-adsorbingresin particle dispersion is prepared.

2.5 wt % gold ion-adsorbing resin particle dispersion (42.4 ml) is thenadded into 1580 ml of pure water, 528 mM aqueous dimethylamine boranesolution (10 ml) is dripped for 2 minutes as agitation is performed at160 rpm under 3° C., agitation is then performed under room temperaturefor 2 hours, and thereby resin-gold composite, the average particle sizeof which is 345 nm, is obtained. The resin-gold composite isprecipitated by centrifugal separation (3100 rpm, 60 minutes),supernatant is removed, the resin-gold composite is dispersed into purewater again, this operation is repeated three times, refining andconcentration regulation are performed by dialysis, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe prepared resin-gold composite determined according to the method is0.96. In addition, the average particle size of the formed goldparticles is 24.6 nm, and the amount of carried gold is 48.5 wt %. Inthe resin-gold composite, the gold particles include encased goldparticles completely encased in the resin particle, partially exposedgold particles having a portion embedded in the resin particle and aportion exposed from the resin particle, and surface-adsorbed goldparticles absorbed on the surface of the resin particle, and at leastportions of the gold particles are three-dimensionally distributed onthe surface section of the resin particle.

<Immunochromatography Evaluation>

The same operation is carried out as in embodiment 1 to prepareresin-gold composite-marked antibody dispersion.

Determination utilizing immunochromatography is carried out by using theprepared resin-gold composite-marked antibody dispersion, and theperformance of the resin-gold composite dispersion is evaluated. Theresult is represented below.

TABLE 11 Slot No. 1 2 3 4 5 6 7 8 9 10 11 12 Antigen dilution APC × APC× APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 2 4 816 32 64 128 256 512 1024 control Coloration After 5 4.0 3.0 2.5 2.0 1.51.0 0.5 0.1 0 0 0 0 level minutes After 10 5.0 4.0 3.5 3.0 2.5 2.0 1.00.5 0.1 0.1 0 0 minutes After 15 6.0 5.0 4.0 3.5 3.0 2.5 1.5 1.0 0.5 0.10 0 minutes

According to Table 11, it can be determined that the resin-goldcomposite-marked antibody shows good coloration for the antigen diluted256 folds.

The determined results of absorbance in 570 nm in above-mentionedembodiment 1 to embodiment 9, comparative example 1 and comparativeexample 2 are gathered and shown in Table 12 and Table 13.

TABLE 12 Em- Em- Em- Em- Em- Em- Em- bodi- bodi- bodi- bodi- bodi- bodi-bodi- ment ment ment ment ment ment ment 1 2 3 4 5 6 7 Absorbance 0.980.96 0.92 1.69 1.01 0.98 0.99 in 570 nm Evaluation ∘ ∘ ∘ ∘ ∘ ∘ ∘

TABLE 13 Embodi- Embodi- Comparative Comparative ment 8 ment 9 Example 1Example 2 Absorbance 1.03 0.96 0.83 1.32 in 570 nm Evaluation ∘ ∘ Δ ∘

Embodiment 10

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 2.00 g) aredissolved into 80 g of pure water, 2-vinylpyridine (2-VP, 9.90 g) anddivinyl benzene (DVB, 0.100 g) are added, and under nitrogen flow,agitation is performed at 250 rpm under 60° C. for 30 minutes. Afteragitation, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA,0.100 g) dissolved in 9.00 g of pure water is dripped for 5 minutes, andis agitated at 250 rpm under 60° C. for 6 hours, and thereby resinparticles, the average particle size of which is 0.45 μm, are obtained.The resin particles are precipitated by centrifugal separation (9000rpm, 10 minutes), supernatant is removed, the resin particles are thendispersed in pure water again, and thereby 10 wt % resin particledispersion is obtained.

<Synthesis of Resin-gold Composite>

30 mM aqueous chloroauric acid solution (80 g) is added into the resinparticle dispersion (3.05 g), and is left alone under room temperaturefor 24 hours. Afterwards, the resin particles are precipitated bycentrifugal separation (3000 rpm, 10 minutes), supernatant is removed,so that redundant chloroauric acid is removed, the resin particles arethen dispersed into 55 g of pure water again, and thereby goldion-adsorbing resin particle dispersion is prepared. After the goldion-adsorbing resin particle dispersion (45 g) is dripped into 10 mMaqueous dimethylamine borane solution (450 ml) for 8 minutes, agitationis performed under room temperature for 2 hours, and thereby aresin-gold composite, the average particle size of which is 0.6 μm, isobtained. The resin-gold composite is precipitated by centrifugalseparation (3000 rpm, 120 minutes), supernatant is removed, theresin-gold composite is then dispersed into 37 g of pure water again, anultrafiltration membrane is used for refining, and thereby 1 wt %resin-gold composite dispersion is obtained. The result of absorbance ofthe resin-metal composite in the resin-gold composite dispersiondetermined according to the method is 1.20. In addition, the averageparticle size of the gold particles in the resin-gold composite obtainedin this way is 22.0 nm, and the amount of carried gold is 49.4 wt %. Thescanning electron microscope (SEM) picture of the obtained resin-goldcomposite is shown in FIG. 8.

Embodiment 11

Besides adding 10 mM aqueous chloroauric acid solution (56 g) into theresin particle dispersion (3.05 g) obtained in embodiment 10, by thesame method as embodiment 10, gold ion-adsorbing resin particledispersion, a resin-gold composite (average particle size: 0.6 μm) and 1wt % resin-gold composite dispersion are obtained. The absorbance of theresin-gold composite in the resin-gold composite dispersion is 1.04. Inaddition, the average particle size of the gold particles in theresin-gold composite in the resin-gold composite dispersion obtained inthis way is 7.61 nm, and the amount of carried gold is 36.8 wt %.

Embodiment 12

<Synthesis of Resin Particles>

2-VP (9.90 g) and DVB (0.200 g) are added into 450 g of pure water, andunder nitrogen flow, agitation is performed at 250 rpm under 60° C. for30 minutes. After 30 minutes of agitation, AIBA (0.100 g) dissolved in9.00 g of pure water is dripped for 5 minutes, agitation is performed at250 rpm for 6 hours, and thereby resin particles, the average particlesize of which is 0.10 μm, are obtained. The resin particles areprecipitated by centrifugal separation (9000 rpm, 20 minutes),supernatant is removed, the resin particles are then dispersed in purewater again, and thereby 10 wt % resin particle dispersion is obtained.

<Synthesis of Resin-gold Composite>

30 mM aqueous chloroauric acid solution (1.0 g) is added into the resinparticle dispersion (0.1 g), and is left alone under room temperaturefor 24 hours. Afterwards, the resin particles are precipitated bycentrifugal separation (3000 rpm, 10 minutes), supernatant is removed,so that redundant chloroauric acid is removed, the resin particles arethen dispersed into 1.5 g of pure water again, and thereby goldion-adsorbing resin particle dispersion is prepared. After the goldion-adsorbing resin particle dispersion (1.5 g) is dripped into 10 mMaqueous dimethylamine borane solution (65 ml) for 2 minutes, agitationis performed under room temperature for 2 hours, and thereby aresin-gold composite, the average particle size of which is 0.12 μm, isobtained. After 10 wt % dispersion (BYK194) (600 μl) is added into theresin-gold composite and agitated for 1 hour, precipitation is performedby centrifugal separation (9000 rpm, 10 minutes), and supernatant isremoved. Afterwards, an appropriate amount of pure water is added fordispersion again, an ultrafiltration membrane is used for refining, andthereby 1 wt % resin-gold composite dispersion is obtained. Theabsorbance of the resin-metal composite in the resin-gold compositedispersion obtained in this way is 0.84. In addition, the averageparticle size of the gold particles in the resin-gold composite in theobtained resin-gold composite dispersion is 8.80 nm, and the amount ofcarried gold is 28.8 wt %.

Embodiment 13

30 mM aqueous chloroauric acid solution (72.4 g) is added into the resinparticle dispersion (0.33 g) obtained in embodiment 12, and is leftalone under room temperature for 24 hours. Afterwards, the resinparticles are precipitated by centrifugal separation (3000 rpm, 10minutes), supernatant is removed, so that redundant chloroauric acid isremoved, the resin particles are then dispersed into 20 g of pure wateragain, and thereby gold ion-adsorbing resin particle dispersion isprepared. After the gold ion-adsorbing resin particle dispersion (20 g)is dripped into 10 mM aqueous dimethylamine borane solution (200 ml) for7 minutes, agitation is performed under room temperature for 2 hours,and thereby a resin-gold composite, the average particle size of whichis 0.12 μm, is obtained. After 10 wt % dispersion (BYK194) (600 μl) isadded into the resin-gold composite and agitated for 1 hour,precipitation is performed by centrifugal separation (9000 rpm, 10minutes), and supernatant is removed. Afterwards, an appropriate amountof pure water is added for dispersion again, an ultrafiltration membraneis used for refining, and thereby 1 wt % resin-gold composite dispersionis obtained. The absorbance of the resin-metal composite in theresin-gold composite dispersion obtained in this way is 1.06. Inaddition, the average particle size of the gold particles in theresin-gold composite in the obtained resin-gold composite dispersion is7.90 nm, and the amount of carried gold is 35.9 wt %.

Embodiment 14

<Synthesis of Resin Particles>

Besides adding 0.100 g of DVB, by the same method as embodiment 12,resin particles (average particle size: 0.20 μm) and 10 wt % resinparticle dispersion are obtained.

<Synthesis of Resin-gold Composite>

Besides adding 30 mM aqueous chloroauric acid solution (132 g) into theresin particle dispersion (5.0 g), by the same method as embodiment 12,gold ion-adsorbing resin particle dispersion, a resin-gold composite(average particle size: 0.21 μm) and 1 wt % of resin-gold compositedispersion are obtained. The absorbance of the resin-gold composite inthe resin-gold composite dispersion obtained in this way is 1.10. Inaddition, the average particle size of the gold particles in theresin-gold composite in the obtained resin-gold composite dispersion is20.0 nm, and the amount of carried gold is 36.0 wt %.

Embodiment 15

<Synthesis of Resin-gold Composite>

Besides adding 30 mM aqueous chloroauric acid solution (198 g) into theresin particle dispersion (5.0 g) obtained in embodiment 14, by the samemethod as embodiment 12, gold ion-adsorbing resin particle dispersion, aresin-gold composite (average particle size: 0.22 μm) and 1 wt %resin-gold composite dispersion are obtained. The absorbance of theresin-metal composite in the resin-gold composite dispersion obtained inthis way is 1.12. In addition, the average particle size of the goldparticles in the resin-gold composite in the obtained resin-goldcomposite dispersion is 22.6 nm, and the amount of carried gold is 37.0wt %.

The determined results of absorbance in 570 nm in above-mentionedembodiment 10 to embodiment 15 and comparative example 1 are gatheredand shown in Table 14.

TABLE 14 Em- Em- Em- Em- Em- Em- Compar- bodi- bodi- bodi- bodi- bodi-bodi- ative ment ment ment ment ment ment Exam- 10 11 12 13 14 15 ple 1Absorbance 1.20 1.04 0.84 1.06 1.10 1.12 0.83 in 570 nm Evaluation ∘ ∘ Δ∘ ∘ ∘ Δ

Experimental Examples Related to Preparation of Marked AntibodyPreparation Example 1

<Synthesis of Resin Particles>

After Aliquat 336 [produced by Aldrich company] (1.00 g) andpoly(ethylene glycol) methyl ether methacrylate (PEGMA, 2.00 g) aredissolved into 80 g of pure water, 2-vinylpyridine (2-VP, 9.90 g) anddivinyl benzene (DVB, 0.100 g) are added, and under nitrogen flow,agitation is performed at 250 rpm under 60° C. for 30 minutes. Afteragitation, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AIBA,0.100 g) dissolved in 9.00 g of pure water is dripped for 5 minutes, andis agitated at 250 rpm under 60° C. for 6 hours, and thereby resinparticles A-1, the average particle size of which is 0.36 μm, areobtained. A-1 is precipitated by centrifugal separation (9000 rpm, 10minutes), supernatant is removed, the resin particles are then dispersedin pure water again, and thereby 2.1 wt % of resin particle dispersionB-1 is obtained.

<Synthesis of Resin-Gold Composite>

30 mM aqueous chloroauric acid solution (106.6 g) is added into B-1(19.09 g), and is left alone under room temperature for 24 hours.Afterwards, the resin particles are precipitated by centrifugalseparation (3000 rpm, 10 minutes), supernatant is removed, so thatredundant chloroauric acid is removed, the resin particles are thendispersed into 40 g of pure water again, and thereby gold ion-adsorbingresin particle dispersion C-1 is prepared. After C-1 (20 g) is drippedinto 3.3 mM aqueous dimethylamine borane solution (600 ml) for 4minutes, agitation is performed under 8° C. for 1 hour, and is thenperformed under room temperature for 5 hours, and thereby a resin-goldcomposite D-1, the average particle size of which is 0.38 μm, isobtained. D-1 is precipitated by centrifugal separation (3000 rpm, 120minutes), supernatant is removed, an appropriate amount of pure water isthen added for dispersion again, an ultrafiltration membrane is thenused for refining, and thereby 1 wt % resin-gold composite dispersionE-1 is obtained. The result of absorbance of the resin-gold compositeF-1 in E-1 determined according to the method is 1.0. In addition, theaverage particle size of the gold particles in F-1 is 22.0 nm, and theamount of carried gold is 49.1 wt %. In the resin-gold composite, thegold particles include encased gold particles completely encased in theresin particle, partially exposed gold particles having a portionembedded in the resin particle and a portion exposed from the resinparticle, and surface-adsorbed gold particles absorbed on the surface ofthe resin particle, and at least portions of the gold particles arethree-dimensionally distributed on the surface section of the resinparticle.

[Reagents]

In the experimental examples and reference experimental examples, thefollowing reagents and so on are used.

Anti-influenza A monoclonal antibody (7.15 g/mL/PBS): Produced by AdtecCo., Ltd.

Binding buffer a: 100 mM Utilizing HCl to regulate boric acid solutioninto pH≈3

Binding buffer b: 100 mM Utilizing HCl to regulate boric acid solutioninto pH≈4

Binding buffer c: 100 mM Utilizing HCl to regulate boric acid solutioninto pH=5

Binding buffer d: 100 mM Boric acid solution pH≈6.5 Binding buffer e:100 mM Utilizing NaCl to regulate boric acid solution into pH≈7.5.

Binding buffer f: 100 mM Utilizing NaCl to regulate boric acid solutioninto pH≈8.5.

Binding buffer g: 50 mM 2-morpholinoethanesulfonic acid solution pH≈3.8Blocking buffer a: Utilizing HCl to regulate 1 wt % bovine serum albuminsolution into pH≈5.

Blocking buffer b: Utilizing HCl to regulate 1 wt % bovine serum albuminsolution into pH≈7.

Blocking buffer c: Utilizing HCl to regulate 1 wt % bovine serum albuminsolution into pH≈8.5.

Blocking buffer d: Utilizing HCl to regulate 1 wt % bovine serum albuminsolution into pH≈9.5.

Cleaning buffer: Utilizing HCl to regulate 5 mMTris(hydroxymethyl)aminomethane solution into pH≈8.5.

Storage buffer: Adding sucrose into cleaning buffer until concentrationis changed into 10 wt %.

Influenza A positive control (APC): Prepared by using sample treatmentsolution (produced by Adtec Co., Ltd.) to dilute an influenza A viruspassivation antigen (produced by Adtec Co., Ltd.) 100 folds. The antigenconcentration of the APC is equivalent to 5000 FFU/ml.

Negative control: Sample treatment solution (produced by Adtec Co.,Ltd.)

AuNCP beads: Resin-gold composite (t %; average particle size: 380 nm)obtained in preparation example 1

Experimental Example 1

(Binding Step)

0.1 mL of AuNCP beads as a resin-metal composite are put into microtubes[IBIS (registered trademark; produced by AS ONE company) 2 mL], and 0.9mL of binding buffer a is added. After upside-down mixing for sufficientmixing, 100 μg of anti-influenza A monoclonal antibody 100 is added,moreover, upside-down agitation is performed under room temperature for3 hours, and thereby marked antibody-containing solution A-1 whichcontains the anti-influenza A monoclonal antibody marked by utilizingthe resin-metal composite is obtained.

(Blocking Step)

Afterwards, after the marked antibody-containing solution A-1 is cooledby ice bath, centrifugal separation is performed at 12000 rpm for 5minutes, supernatant is removed, 1 mL of blocking buffer a is then addedinto solid component residue, ultrasonic dispersion treatment isperformed for 10 to 20 seconds, further, upside-down agitation isperformed under room temperature for 2 hours, and thereby markedantibody-containing solution B-1 is obtained.

(Cleaning Treatment)

Afterwards, after the marked antibody-containing solution B-1 is cooledby ice bath, centrifugal separation is performed at 12000 rpm for 5minutes, supernatant is removed, 1 mL of cleaning buffer is then addedinto solid component residue, and ultrasonic dispersion treatment isperformed for 10 to 20 seconds. This operation is repeated three timesas cleaning treatment.

(Storage Treatment)

Afterwards, after ice bath cooling, centrifugal separation is performedat 12000 rpm for 5 minutes, supernatant is removed, 1 mL of storagebuffer is then added into solid component residue, ultrasonic dispersiontreatment is performed for 10 to 20 seconds, and thereby markedantibody-containing solution C-1 is obtained.

Experimental Example 2

Binding buffer b is used to substitute for binding buffer a in thebinding step of experimental example 1, and besides, markedantibody-containing solution A-2, marked antibody-containing solutionB-2 and marked antibody-containing solution C-2 are obtained in the sameway as experimental example 1.

Experimental Example 3

Binding buffer c is used to substitute for binding buffer a in thebinding step of experimental example 1, and besides, markedantibody-containing solution A-3, marked antibody-containing solutionB-3 and marked antibody-containing solution C-3 are obtained in the sameway as experimental example 1.

Experimental Example 4

Binding buffer d is used to substitute for binding buffer a in thebinding step of experimental example 1, and besides, markedantibody-containing solution A-4, marked antibody-containing solutionB-4 and marked antibody-containing solution C-4 are obtained in the sameway as experimental example 1.

Reference Experimental Example 1

When binding buffer e is used to substitute for binding buffer a in thebinding step of experimental example 1, resin-metal composite isaggregated, so it is hard to obtain marked antibody-containing solution.

Reference Experimental Example 2

When binding buffer f is used to substitute for binding buffer a in thebinding step of experimental example 1, a resin-metal composite isaggregated, so it is hard to obtain marked antibody-containing solution.

Experimental Example 5

Blocking buffer b is used to substitute for the blocking buffer a in theblocking step of experimental example 1, and besides, markedantibody-containing solution B-5 and marked antibody-containing solutionC-5 are obtained in the same way as experimental example 1.

Experimental Example 6

Blocking buffer c is used to substitute for the blocking buffer a in theblocking step of experimental example 1, and besides, markedantibody-containing solution B-6 and marked antibody-containing solutionC-6 are obtained in the same way as experimental example 1.

Reference Experimental Example 3

As the result of using blocking buffer d to substitute for the blockingbuffer a in the blocking step of experimental example 1, the markedantibody after the binding step shows good dispersibility, however,after the blocking step, marked antibody is aggregated, so it is hard toobtain marked antibody-containing solution.

Experimental Example 7

Binding buffer g is used to substitute for binding buffer a in thebinding step of experimental example 1, and besides, markedantibody-containing solution A-7, marked antibody-containing solutionB-7 and marked antibody-containing solution C-7 are obtained in the sameway as experimental example 1.

<Evaluation Method>

Evaluation is carried out by using the monochrome screen for influenza Aevaluation (produced by Adtec company), and coloration levels after 5minutes, 10 minutes and 15 minutes are compared. A color sample forcolloidal gold determination (produced by Adtec company) is used tomeasure the coloration levels. In screening and evaluation, antigen usesan influenza A positive control (APC). In performance evaluation,antigen uses 2-fold dilution (1- to 1024-fold dilution) of the APC.

<Screening and Evaluation>

3 μL of marked antibody-containing solution C-1, 34 of markedantibody-containing solution C-2, 34 of marked antibody-containingsolution C-3, 34 of marked antibody-containing solution C-4, 34 ofmarked antibody-containing solution C-5, 34 of markedantibody-containing solution C-6 and 34 of marked antibody-containingsolution C-7 which are obtained in experimental example 1 toexperimental example 7 are respectively added into seven pores of the96-well plate, and 1004 of APC is mixed into each well. Afterwards, 504is added into the monochrome screen for influenza A evaluation, andcoloration levels after 5 minutes, 10 minutes and 15 minutes areevaluated. The result is shown in Table 15. Moreover, the higher thevalues in Table 15 are, the higher coloration levels are (intensercoloration).

TABLE 15 Marked antibody-containing solution C-1 C-2 C-3 C-4 C-5 C-6 C-7Coloration After 5 6.5 6.0 6.0 5.5 5.0 4.0 4.0 level minutes After 107.0 6.0 6.0 6.0 6.5 5.5 5.0 minutes After 15 7.0 6.5 7.0 6.5 7.0 6.0 5.0minutes

According to Table 15, it can be determined that the markedantibody-containing solution C-1 obtained in experimental example 1shows the strongest coloration, having excellent marking performance.

<Performance Evaluation>

3 μL of marked antibody-containing solution C-1 obtained in experimentalexample 1 is added into each of 12 wells of the 96-well plate, and 100μL of 2-fold dilution (1- to 1024-fold dilution, respectivelyrepresented as APC×1 to APC×1024) of the APC and 100 μL of negativecontrol are mixed. Afterwards, 504 is added into the monochrome screenfor influenza A evaluation, and coloration levels after 5 minutes, 10minutes and 15 minutes are evaluated. The result is shown in Table 16.Moreover, the higher the values in Table 16 are, the higher colorationlevels are (intenser coloration).

TABLE 16 Marked antibody-containing solution C-1 Antigen dilution APC ×APC × APC × APC × APC × APC × APC × APC × APC × APC × APC × Negative 1 24 8 16 32 64 128 256 512 1024 control Coloration After 5 6.0 5.0 4.0 3.53.0 1.5 1.0 0.5 0.1 0 0 0 level minutes After 10 7.0 6.0 5.0 4.5 4.0 3.02.0 1.0 0.5 0 0 0 minutes After 15 7.5 6.5 5.5 5.0 4.5 3.5 2.5 1.5 1.00.1 0 0 minutes

According to Table 16, it can be determined that the markedantibody-containing solution C-1 obtained in experimental example 1shows good coloration for the antigen diluted 256 folds, havingexcellent marking performance.

The embodiments of the present invention are elaborated above with theillustrated purposes, however, the present invention is not limited bythe embodiments.

The present international application claims the priority of JapanesePatent Application No. 2014-136355 applied on Jul. 1, 2014, and all thecontent of the present application is integrated into this case.

LIST OF REFERENCE NUMERALS

-   10: Resin particle-   20: Metal particle-   30: Encased metal particle-   40: Partially exposed metal particle-   50: Surface-adsorbed metal particle-   60: Surface section-   100: Resin-metal composite-   110: Membrane-   120: Sample addition portion-   130: Determination portion-   131: Capturing ligand-   140: Liquid-absorbing portion-   150: marked antibody-   160: Analyte-   170: Composite-   200: Test strip

1. A resin-metal composite, comprising a structure formed byimmobilizing a plurality of metal particles on a resin particle,characterized in that the plurality of metal particles includes: firstparticles, having a portion exposed from the resin particle; and secondparticles, completely encased in the resin particle; and among the firstparticles and the second particles, at least portions of the particlesare three-dimensionally distributed in a surface section of the resinparticle, and 60 wt % to 100 wt % of the metal particles exist in thesurface section.
 2. The resin-metal composite according to claim 1,wherein relative to the weight of the resin-metal composite, thequantity of the carried metal particles is within a range from 5 wt % to70 wt %.
 3. The resin-metal composite according to claim 1, wherein 5 wt% to 90 wt % of the metal particles existing in the surface section arethe first particles.
 4. The resin-metal composite according to claim 1,wherein the resin particle is a polymer particle which is provided witha substituent group capable of adsorbing metal ions in the structure. 5.The resin-metal composite according to claim 1, wherein the averageparticle size of the metal particles is within a range from 1 nm to 80nm.
 6. The resin-metal composite according to claim 1, the averageparticle size thereof is within a range from 100 nm to 1000 nm.
 7. Theresin-metal composite according to claim 1, wherein the metal particlesare gold particles.
 8. A marker, comprising the resin-metal compositeaccording to claim
 1. 9. The marker according to claim 8, used inadsorbing an antigen or an antibody on the surface of the resin-metalcomposite.
 10. An immunoassay method, characterized by using the markeraccording to claim
 8. 11. A immunoassay reagent, comprising theresin-metal composite according to claim
 1. 12. A method for measuringanalyte, assaying or quantifying an analyte contained in a sample,characterized by using a lateral-flow chromatographic test stripcomprising a membrane and a determination portion formed by immobilizingcapturing ligands specifically bound with the analyte on the membrane tocarry out steps including step (I) to step (III) hereinafter: step (I):step of making the analyte contained in the sample contact with a markedantibody formed by utilizing the resin-metal composite according toclaim 1 to mark an antibody specifically bound with the analyte; step(II): step of making the composite containing the analyte and the markedantibody formed in step (I) contact with the capturing ligands in thedetermination portion; and step (III): step of determining coloredintensity derived from the localized surface plasmon resonance of theresin-metal composite.
 13. An analyte measurement kit, using alateral-flow chromatographic test strip, used to assay or quantify ananalyte contained in a sample, and the analyte measurement kitcomprising: the lateral-flow chromatographic test strip, comprising amembrane and a determination portion formed by immobilizing capturingligands specifically bound with the analyte on the membrane; and anassay reagent, containing a marked antibody formed by utilizing theresin-metal composite according to claim 1 to mark an antibodyspecifically bound with the analyte.
 14. A lateral-flow chromatographictest strip, used to assay or quantify an analyte contained in a sample,comprising a membrane; a determination portion, formed by immobilizingcapturing ligands specifically bound with the analyte on the membrane inthe spreading direction of a sample; and a reaction portion, comprisinga marked antibody formed by utilizing the resin-metal compositeaccording to claim 1 to mark an antibody specifically bound with theanalyte further upstream than the determination portion.